This proposal will study warm-type IgG red cell (RBC) autoantibodies (AAb) directed against blood group related determinants on band 3 and glycophorin, since over 90% of AAbs fail to react with Rh null or En(a-) RBC. Peptide mapping studies from this laboratory have recently confirmed the association of band 3 with the Rh complex and others have demonstrated the absence of glycophorin A in En(a-) cells. Both Band 3 and glycophorin A are the two main surface exposed RBC membrane proteins. Advances in RBC membrane biochemistry, in hybridoma technology and a theoretical framework for RBC autoimmunity based on the idiotypic network theory will facilitate the proposed studies. Most importantly, the project will use a recently developed method from this laboratory which permits the isolation, purification and radiolabeling of human RBC AAb's in the absence of non-specific binding behavior. Five groups of studies will be carried out. 1) Studies on the interaction of radiolabeled AAbs with RBC will serve to characterize quantitatively AAb blood group specificity, site densities, kinetic constants, thermodynamic values, and other properties of the AAb. 2) The idiotypic relationships between RBC autoantibodies and Rh alloantibodies will be studied using both polyclonal and monoclonal anti-idiotypic antibodies to determine AAb and allo-antibody cross-reactivity and to test whether anti-idiotypic antibody and antigen share epitopes. 3) Studies with drugs associated with RBC autoimmunity (aldomet and others) will test their ability to alter or modify idiotope anti-idiotope interactions of AAbs and alloantibodies. 4) Studies on the cell surface topology of AAb determinants using immunoelectron microscopy and AAbs labeled with protein A-colloidal gold to determine surface spatial relationships within AAb and between alloantibodies and to investigate other phenomena (C fixation, agglutination) dependent on antibody location. 5) Studies to identify in vitro predictors of the hemolytic potential of AAbs and on the influence of cytoskeletal alterations on AAb binding in RBC of patients with hereditary hemolytic anemias due to cytoskeletal defects. The proposed studies should increase our understanding of the molecular basis of red cell autoimmunity and possibly lead to a more effective clinical management of patients with AIHA.